27. Prepare a PCR master mix by combining the following reagents in the order shown:
162 µL Sterile RNAse-free water 20 µL 10X Encyclo buffer
4 µL 50X dNTP mix (10 mM each) 6 µL PCR primer M1 (10 µM) 4 µL 50X Encyclo polymerase mix 196 µL Total volume
I Note: If you normalize several cDNA samples, increase the volume of PCR master mix accordingly.
28. Mix the contents by gently flicking the tubes. Spin the tubes briefly in a microcentrifuge.
29. Aliquot 1 µL of each sample from step 26 into an appropriately la-beled sterile 0.2 mL PCR tubes.
30. Add 49 µL of the PCR master mix into the tubes.
31. Mix contents by gently flicking the tubes. Spin the tubes briefly in a microcentrifuge.
32. If the thermal cycler used is not equipped with a heated lid, overlay each reaction with a drop of mineral oil. Close the tubes, and place them into a thermal cycler.
33. Subject the tubes to PCR cycling using the following program:
Initial denaturation 95°C 1 min
Cycling 7 cycles 95°C 15 sec
66°C 20 sec 72°C 3 min
34. Put the Experimental tubes on ice. Use the Control tube to deter-mine the optimal number of PCR cycles, as follows:
(a) Aliquot 12 µL from the seven-cycle control tube into a clean mic-rocentrifuge tube (for agarose/EtBr gel analysis).
(b) Run two additional cycles (for a total of nine cycles) with the re-maining 38 µL of the control PCR mixture.
(c) Aliquot 12 µL from the nine-cycle control tube into a clean mic-rocentrifuge tube.
(d) Run two additional cycles (for a total of 11) with the remaining 26 µL of the control PCR mixture.
(e) Aliquot 12 µL from the 11-cycle control tube into a clean micro-centrifuge tube.
(f) Run two additional cycles (for a total of 13) with the remaining 14 µL of the control PCR mixture.
35. Analyze 5 µL aliquots of each control PCR reaction (seven, nine, eleven, and thirteen cycles; from step 34) alongside 0.1 µg of 1 kb DNA size marker on a 1.5% (w/v) agarose/EtBr gel, run in 1X TAE buffer. Store the remaining materials on ice.
36. Determine the optimal number of cycles required for amplification of the control cDNA, as follows:
PCR cycles
7 9 11 13
M 1 2 3 4
1.0 kb 1.5 kb 3.0 kb
Fig. 6. Analysis for optimizing PCR parameters.
5 µl of each aliquot from the Control tube (from step 34) was electrophoresed on a 1.5%
agarose/EtBr gel in 1X TAE buffer following the indicated number of PCR cycles. The optimal number of cycles determined in this experiment was 9. Lane M: 1-kb DNA ladder size markers, 0.1 µg loaded.
When the PCR product yield stops increasing with an additional cy-cle, the reaction has reached a plateau. The optimal number of cycles should be one or two cycles less than that needed to reach the plateau.
A typical electrophoresis result indicating an optimal number of PCR cycles should appear as a moderately strong cDNA smear of the expected size distribution, with several bright bands corre-sponding to abundant transcripts. For cDNA prepared from most mammalian RNAs, the overall signal intensity (relative to that of 0.1 µg of 1 kb DNA size marker run on the same gel) should be roughly similar to that shown in lane 2 of Fig. 6.
If the cDNA smear appears in the high-molecular-weight region of the gel (e.g., as shown in lane 4 of Fig. 6), especially if no bright bands are distinguishable, your PCR parameters may be subopti-mal. If the smear is faint, such as that shown in lane 1 of Fig. 6, this indicates that too few PCR cycles were used for amplification (see «Troubleshooting», subsection XI. B).
I Note: The optimal number of PCR cycles must be determined indi
vidually for each experimental sample. Using the optimal number of PCR cycles ensures that the ds cDNA remains in the exponential phase of amplification. PCR overcycling is extremely undesirable, as it yields nonspecific PCR products. Therefore, it is better to use fewer cycles than too many.
37. Retrieve the seven-cycle experimental tubes from ice, return them to the thermal cycler, and (if necessary) subject them to additional PCR cycles to reach the optimal number indicated in the control cDNA experiment. Next, immediately subject the tubes to an addi-tional nine cycles of PCR.
I Note: In total, the experimental tubes should be subjected to X+9 PCR cycles, where X is the optimal number of PCR cycles determined for the control tube. In the example shown in Fig. 6, the optimal num
ber of PCR cycles determined using the control tube is nine. Thus, in this example X=9, and the seven-cycle experimental tubes should be subjected to 2+9 additional PCR cycles.
38. Analyze 5 µL of each experimental PCR reaction alongside 5 µL of the control PCR reaction representing the optimal number of PCR cycles, and 0.1 µg of 1 kb DNA size marker on a 1.5% (w/v) agarose/EtBr gel run in 1X TAE buffer.
39. Store remaining control cDNA representing the optimal number of PCR cycles at -20°C.
40. Compare the banding pattern intensity of the PCR products from the experimental and control tubes, as follows:
• If the overall signal intensity of PCR products from the experimen-tal tubes is similar to that of the control, proceed to step 41.
• If the smear from the experimental tubes is much fainter than that of the control, PCR undercycling may be an issue. Sub-ject the experimental tubes to two or three more PCR cycles and repeat the electrophoresis. If there is still a strong difference between the overall signal intensity of all experimental PCR prod-ucts and the control, the normalization process might have been too strong. (see «Troubleshooting», subsection XI. C).
• If the overall signal intensity of the experimental PCR products is much stronger than the control, especially if there are distinct bright bands present, the normalization process might have been unsuccessful (see «Troubleshooting», subsection XI. C).
41. Select the tube(s) showing efficient normalization. For comparison, Fig. 7 shows a characteristic gel profile of normalized human cDNA.
A typical result, indicative of efficient normalization, should have the following characteristics:
• PCR products from experimental tube(s) containing efficiently normalized cDNA appear as a smear without clear bands, whereas those from the non-normalized controls usually present a number of distinct bands.
• The average length of the efficiently normalized cDNA is congru-ous with the average length of cDNA from the non-normalized control tube.
I Note: The upper boundary of the normalized cDNA smear usu
ally does not exceed 5 kb. If the normalized cDNA appears as a uniform smear stretching from the input well to the low-molecu
lar-weight region or bands are visible in the normalized cDNA sample, see «Troubleshooting», subsection XI. D.
42. If cDNA from two or more Experimental tubes (step 41) appears well normalized, combine contents of these tubes in one sterile 1.5 mL tube, mix well by vortexing and spin the tube briefly in a microcent-rifuge.
Now you have obtained normalized ds cDNA. The resulting amount of ds cDNA per reaction with total volume of 50 µL is anticipated to be in a range of 0.75 -1.35 µg. This normalized cDNA can be stored at -20 ℃ up to one month and used afterwards to prepare more normalized
cDNA.
43. Please refer to Section IX "Recommendations for further process-ing of normalized cDNA" to choose the protocol for further pro-cessing of amplified ds cDNA before use in intended downstream applications.
I Note: Before cDNA processing you can estimate normalization effi
ciency using quantitative PCR or Virtual Northern blot with marker genes of known abundance. Please refer to the Section VIII "Analysis of normalization efficiency".
M 1 2 3 4
Fig. 7. Analysis of cDNA normalization results 5-µl aliquots of the PCR products were loaded on a 1.5% agarose/EtBr gel. Lane M: 1-kb DNA size mark-ers, 0.1 µg loaded. Lane 1: cDNA from the Control tube. Lane 2: cDNA from the S1_DSN1/4 tube. Lane 3: cDNA from the S1_DSN1/2 tube. Lane 4: cDNA from the S1_DSN1 tube. In this experiment efficient normalization was achieved in the S1_DSN1/2 tube (lane 3). In the S1_DSN1/4 tube (lane 1) normaliza-tion was not completed, in the S1_DSN1 tube (lane 4) DSN treatment was excessive, resulting in partial cDNA degradation.
VIII Analysis of normalization efficiency
cDNA normalization considerably decreases the concentrations of highly abundant transcripts in a cDNA population (by about 1000-fold for the most abundant transcripts), but typically doesn’t change the concentrations of medium abundance transcripts. The concentrations of rare molecules may slightly increase, or they may remain the same.
As a result, the normalized cDNA is enriched for rare transcripts, but also includes medium and high abundance transcripts.
Either quantitative PCR (qPCR) or virtual Northern blot [8] can be used to estimate the efficiency of normalization prior to cDNA cloning or sequencing. Comparing the abundance levels of already studied tran-scripts before and after normalization, one can see a relative reduction in the representation level of abundant transcripts in a normalized cDNA sample (in comparison with a non-normalized one).
Alternatively, clones may be randomly picked and sequenced from nor-malized and non-nornor-malized cDNA libraries, and the gene discovery rates may be compared between the libraries. A successfully nor-malized cDNA library will have a higher gene discovery rate than a non-normalized library; however, the particular characteristics of a given library will depend on the initial cDNA redundancy, the cDNA GC-content, the number of clones tested, etc.
I Some problems that may occur during an analysis of normalization efficiency are discussed in the "Troubleshooting", subsection XI. E).
Analysis of normalization efficiency by qPCR
Important notes:I Trimmer-2 kit provides GAPD primer mix allowing qPCR-testing of nor
malization efficacy in human and mouse cDNA samples on the example of glyceraldehyde-3-phosphate dehydrogenase (GAPD) that is expressed at high levels in most mammalian tissues and cell lines.
For cDNA from other sources (non-human and non-mouse), please select and design primers specific for source-specific high abundance transcripts.
Reagents required
• Ready-to-use qPCR Master Mix containing SYBR Green I dye and ROX reference dye
qPCR protocol
1. Make sure that the difference between concentrations of normal-ized cDNA (from step 42 of the Normalization protocol) and control cDNA (from step 39 of the Normalization protocol) does not exceed 2-times. If necessary, equalize cDNA concentrations.
I Note: cDNA concentration should be estimated using agarose gel-elec
trophoresis or spectrophotometric analysis.
2. Aliquot 1 µL of normalized cDNA (from step 42 of the Normalization protocol) into a sterile 1.5 mL tube; add 39 µL of sterile RNAse-free water to the tube, mix well by vortexing and spin the tubes briefly in a microcentrifuge.
I Note: If the normalized cDNA sample was stored at -20 ℃, pre-heat it at 65℃ for 1 min and mix by gently flicking the tubes before taking aliquots. Store the remaining cDNA at -20 ℃.
3. Aliquot 1 µL of control cDNA (from step 39 of the Normalization protocol) into another sterile 1.5 mL tube; add 39 µL of sterile RNAse-free water to the tube, mix well by vortexing and spin the tubes briefly in a microcentrifuge.
I Note: If the control cDNA sample was stored at -20 ℃, pre-heat it at 65℃ for 1 min and mix by gently flicking the tubes before taking aliquots. Store the remaining cDNA at -20 ℃.
4. Prepare qPCR reactions with primers specific for high abundance transcripts in the experimental cDNA samples and 1 µL aliquots of the diluted cDNA from steps 2 and 3.
5. Perform PCR cycling as described in the instructions (or instruction booklet) provided with the ready-to-use qPCR Master Mix. Three--step cycling protocol is recommended.
I Note: Appropriate annealing temperature for the GAPD primer mix provided in the Trimmer-2 kit is 60℃.
6. When cycling is completed, use thermal cycler software to identify Ct for each PCR reaction. Calculate mean value Ct for each cDNA sample. Mean Ct for the control cDNA should be less than 20.
I Note: If mean Ct for the control cDNA is 21 or more, this indicates that the transcript tested is not in high abundance in the cDNA sample.
Thus, its concentration may remain unchanged during normalization.
In this case, repeat qPCR with primer pair specific to another high abundance transcript.
I Note: GAPD is expressed at high levels in most human and mouse tissues and cell lines, however there could be some exceptions. In some samples, GAPD transcripts belong to intermediate or low abundance groups, and unchanged or slightly increased concentration of these tran
scripts in normalized cDNA is observed. In this case, please select other marker genes that are abundant in samples of interest to test normali
zation efficiency.
7. Calculate ΔCt as follows:
ΔCt = CtC – CtN,
where CtCis the mean Ct for the control cDNA sample and CtNis the mean Ct for the normalized cDNA sample.
ΔCt ≥ 5 indicates effective normalization. ΔCt ≤ 4 indicates un-successful normalization.
-0.1
Fig. 8. Analysis of cDNA normalization results by qPCR. Efficiency of normalization of human and mouse brain cDNA was tested using quantitative PCR with GAPD primer mix.
ΔCt = 9 indicates successful normalization in both cases.
IX Recommendations for further processing of normalized cDNA
Adapter pair used for ds cDNA preparation
Intended application Recommendations
PlugOligo-1 and CDS-1 adapters OR
SMARTer II A Oligonucleotide and 3’ SMART CDS Primer II A
Non-directional cDNA
SMART IV Oligonucleotide and CDS-4M adapter
PlugOligo-1 and CDS-Gsu adapters OR
SMARTer II A Oligonucleotide and CDS-Gsu adapter
Roche/454 sequencing
see Appendix F
Appendix A cDNA synthesis and amplification using SMART-based kit (Clontech)
Reagents required
• Purified RNA for cDNA synthesis (at least 1-2 µg of total RNA or 0.5-1 µg of polyA+ RNA)
I The RNA may be isolated using a number of suitable methods that yield stable RNA preparations from most biological sources; two examples are the TRIzol method (Gibco/Life Technologies) and the RNeasy kit (Qia
gen). Total RNA can also be isolated as described in [9].
Following RNA isolation, RNA quality should be estimated using dena
turing formaldehyde/agarose gel electrophoresis, as described by Sam
brook [10]. The RNA length generally depends on the RNA source, however, if experimental RNA is not larger than 1.5 kb, we suggest you prepare fresh RNA after checking the quality of the RNA purification reagents. If problems persist, you may need to find another source of tissue/cells.
In general, genomic DNA contamination does not affect cDNA synthesis, meaning that DNase treatment is not required. When necessary, excess genomic DNA can be removed by LiCl precipitation or phenol:chloroform extraction.
• SMART-based kit (Clontech)
I Please refer to the section IV (cDNA preparation) to choose the kit suit
able for your needs.
• Encyclo PCR Kit (Evrogen, Cat.# PK001) or analogues
• QIAquick PCR Purification Kit (Qiagen)
• Sterile molecular biology grade water (sterile RNase-free water)
• Agarose gel electrophoresis reagents
• DNA size markers (1-kb DNA ladder)
First-strand cDNA synthesis Important notes:
I The following protocol describes the use of reagents provided in SMART-based kits (Clontech) and additional 3’-end adapters included in Trimmer-2 kit for synthesis of first-strand cDNA suitable for normaliza
tion procedure and allowing various downstream application of normalized cDNA. Please refer to the Section IV (cDNA preparation) to choose the adapter pair suitable for your needs.
I The sequence complexity and average length of the normalized cDNA library strongly depend on the quality and amount of the starting RNA material used to prepare the cDNA. For best results, at least 1-2 µg of total RNA or 0.5-1 µg of polyA+ RNA should be used at the beginning of first-strand cDNA synthesis. The minimum amount of starting RNA for cDNA synthesis is 250 ng of total RNA or 100 ng of polyA+ RNA.
I We strongly recommend that you perform a positive control cDNA synthesis with control RNA provided in the cDNA synthesis kit, that you use, simul
taneously with experimental cDNA synthesis. This control is performed to verify that all reagents are working properly.
1. Immediately before taking the aliquot for the cDNA synthesis, heat the RNA samples at 65°C for 1-2 min, mix the contents by gently flicking the tube (to prevent RNA aggregation), and then spin the tube briefly in a microcentrifuge.
2. For each RNA sample, combine the following reagents in a sterile PCR tube:
1-3 µL RNA solution in sterile RNase-free water
(1-2µg of total RNA or 0.5-1 µg of polyA(+) RNA)
For the control reaction use 1 µL (1 µg) of the control RNA 1 µL 5’-end adapter*
1 µL 3’-end adapter*
x µL Sterile RNAse-free water 5 µL Total volume
*Refer to Section IV cDNA preparation to choose the adapter pair that can be used with the SMART-based cDNA synthesis kit.
3. Gently pipette the reaction mixtures and spin the tubes briefly in a microcentrifuge. If the thermal cycler used is not equipped with a heated lid, overlay each reaction with a drop of molecular biology-grade mineral oil to prevent the loss of volume due to evaporation.
4. Incubate the mixture in a thermal cycler at 70°C for 2 min (use heated lid).
5. Decrease the incubation temperature to 42°C. Keep the tubes in the thermal cycler at 42°C while preparing the RT master mix (∼1 to 3 min).
6. While steps 4 and 5 are ongoing, prepare an RT master mix for each reaction tube by combining the following reagents in the or-der shown:
2 µL 5X First-strand buffer 1 µL DTT (20 mM)
1 µL 50X dNTP
1 µL SMARTScribe MMLV Reverse Transcriptase 5 µL Total volume
I Note: Optionally, 0.5 µL of RNase inhibitor (20 U/µL) can be added to the reaction to prevent RNA degradation during cDNA synthesis.
7. Gently pipette the RT master mix and spin the tube briefly in a mic-rocentrifuge.
8. Add 5 µL RT master mix to each reaction tube from step 5. Gently pipette the reaction mix, and spin the tubes briefly in a microcentri-fuge to deposit contents at the bottom.
I Note: Do not remove the reaction tubes from the thermal cycler ex
cept for the time necessary to add the RT master mix.
9. Incubate the tubes at 42°C for 1.5 h.
10. After incubation, place the tubes on ice to terminate the first-strand cDNA synthesis.
First-strand cDNA can be stored at -20 ℃ for up to one month and used for ds cDNA amplification.
cDNA amplification
11. For each first-strand cDNA sample from step 10 above, prepare a PCR mixture by combining the following reagents in the order shown:
80 µL Sterile RNase-free water 10 µL 10X Encyclo PCR buffer*
2 µL dNTP mix (10mM each)*
4 µL PCR primer M1 (10 µM)
2 µL First-strand cDNA (from step 10) 2 µL 50X Encyclo polymerase mix*
100 µL Total volume
* The component is provided in the Encyclo PCR kit.
I Note: If the first-strand cDNA samples were stored at -20 ℃, pre-heat them at 65℃ for 1 min, then mix by gently flicking the tubes before taking aliquots. Store the remaining first-strand cDNA at -20 ℃.
12. Mix the contents by gently flicking the tube. Spin the tube briefly in a microcentrifuge.
13. If the thermal cycler used is not equipped with a heated lid, overlay each reaction mixture with a drop of mineral oil. Close the tubes, and place them into a thermal cycler.
14. Subject the tubes to PCR cycling using the following program:
Initial denaturation 95°C 1 min
Cycling X cycles* 95°C 15 sec
66°C 20 sec 72°C 3 min
*X is the optimal number of PCR cycles for a given amount of total or poly(A)+
RNA used for the first-strand cDNA synthesis according to the Table 3 below.
Table 3. PCR cycling parameters
Total RNA (µg) polyA+ RNA (µg) Number of PCR cycles
1.0-1.5 0.5-1.0 13-15
0.5-1.0 0.25-0.5 15-18
0.25-0.5 0.1-0.25 18-21
The recommended parameters were tested using placenta and skeletal muscle total and poly(A)+ RNA and an MJ Research PTC-200 Thermal Cycler. Optimal pa-rameters may vary with different thermal cyclers, polymerase mixes, and templates.
Use the minimal possible number of cycles possible, since overcycling may yield a nonspecific PCR product. If necessary, undercycling can be easily rectified by plac-ing the reaction tube back into the thermal cycler for a few more cycles (see also Troubleshooting Guide, Section B).
Please note, cDNA samples that require more than 25 PCR cycles to be amplified may not be representative. We do not recommend using such samples for normali-zation. Repeat cDNA amplification using larger amounts of first-strand cDNA.
15. When cycling is complete, place the tubes on ice.
16. Analyze 5 µL of the PCR product alongside 0.1 µg of 1 kb DNA size marker on a 1.5% (w/v) agarose/EtBr gel run in 1X TAE buffer to estimate cDNA quality and concentration.
A typical electrophoresis result indicating successful cDNA syn-thesis should appear as a moderately strong cDNA smear of the
A typical electrophoresis result indicating successful cDNA syn-thesis should appear as a moderately strong cDNA smear of the